Altered metabolism is a poorly understood feature of tumors that holds great promise for improved cancer therapy. However, success depends on understanding how metabolic regulation provides an advantage for tumor cells. Our understanding of how cancer cells meet their metabolic needs is based primarily on studies of cultured cells; and nutrient levels in vitro are significantly different from those experienced by tumor cells in vivo. Therefore, better models to study cancer metabolism in vivo are desperately needed. Recently, several studies have converged on serine metabolism as an important metabolic pathway that is dysregulated in cancer. Increased flux through the serine synthesis pathway branching from glycolysis is critical for cancer cell proliferation and tumor growth, and many key metabolic regulatory events promote an increase in new serine synthesis. However, increased serine pathway flux is necessary for some cancer cells even when serine is abundant, and why increased serine metabolism is important for cancer is not understood. The gene encoding the first enzyme of the serine synthesis pathway, PHGDH, is amplified in human tumors, including melanoma, and represents a way cancer cells increase serine production from glucose. Increased carbon flux into serine synthesis can be induced in cells by increasing PHGDH enzyme expression, and this presents the opportunity to determine if more serine synthesis can be a driver of malignancy. It also provides a tool to modulate serine synthesis and study the impact of this pathway on tumor metabolism. To better understand the role of serine synthesis in tumor biology, we propose in Aim 1 to generate a model of PHGDH-amplified cancer. Specifically, we will use a genetically engineered mouse model where PHGDH expression can be controlled in a temporal and tissue-specific manner to determine if increased PHGDH expression to levels found in human tumors promotes tumor initiation. For these experiments we will focus on melanoma, and evaluate the ability of PHGDH to cooperate with other genetic events associated with melanoma in humans. By evaluating tumors that form when PHGDH is expressed, we will also determine whether continued PHGDH expression and increased serine biosynthesis is required for tumor maintenance. In Aim 2, we propose to study how increased serine biosynthesis influences metabolism to promote tumor growth in vivo. We will track the metabolism of stable isotope labeled nutrients in melanomas with and without PHGDH expression to determine how glucose-derived serine is used by these tumors, and understand the impact of increased serine biosynthesis on cancer metabolism. These studies will combine the use of unique animal models with current technology to interrogate metabolic pathway biochemistry and increase the understanding of tumor metabolism in vivo. They will also validate serine synthesis as a cancer drug target and aid efforts to target metabolism in patients.